Yeast Vps13 promotes mitochondrial function and is localized at membrane contact sites.

The Vps13 protein family is highly conserved in eukaryotic cells. Mutations in human VPS13 genes result in a variety of diseases, such as chorea acanthocytosis (ChAc), but the cellular functions of Vps13 proteins are not well defined. In yeast, there is a single VPS13 orthologue, which is required for at least two different processes: protein sorting to the vacuole and sporulation.

Functional expression of human adenine nucleotide translocase 4 in Saccharomyces cerevisiae.

The adenine nucleotide translocase (ANT) mediates the exchange of ADP and ATP across the inner mitochondrial membrane. The human genome encodes multiple ANT isoforms that are expressed in a tissue-specific manner. Recently a novel germ cell-specific member of the ANT family, ANT4 (SLC25A31) was identified.

Hsp90 and mitochondrial proteases Yme1 and Yta10/12 participate in ATP synthase assembly in Saccharomyces cerevisiae.

Hsc82 and Hsp82, the Hsp90 family proteins of yeast, are both required for fermentative growth at 37°C. Inactivation of either of the mitochondrial AAA proteases, Yme1 or Yta10/12, allows fermentative growth of hsc82∆ or hsp82∆ strains at 37°C. Genetic evidence indicates interaction of Hsc82/Hsp82 with the Yme1 and Yta10/Yta12 complexes in promoting F(1)F(o)-ATPase activity, with Hsc82 specifically required for F(1)-ATPase assembly.

The molecular basis for relative physiological functionality of the ADP/ATP carrier isoforms in Saccharomyces cerevisiae.

AAC2 is one of three paralogs encoding mitochondrial ADP/ATP carriers in the yeast Saccharomyces cerevisiae, and because it is required for respiratory growth it has been the most extensively studied. To comparatively examine the relative functionality of Aac1, Aac2, and Aac3 in vivo, the gene encoding each isoform was expressed from the native AAC2 locus in aac1Delta aac3Delta yeast. Compared to Aac2, Aac1 exhibited reduced capacity to support growth of yeast lacking mitochondrial DNA or of yeast lacking the ATP/Mg-P(i) carrier, both conditions requiring ATP import into the mitochondrial matrix through the ADP/ATP carrier.

Mutations in the Atp1p and Atp3p subunits of yeast ATP synthase differentially affect respiration and fermentation in Saccharomyces cerevisiae.

ATP1-111, a suppressor of the slow-growth phenotype of yme1Delta lacking mitochondrial DNA is due to the substitution of phenylalanine for valine at position 111 of the alpha-subunit of mitochondrial ATP synthase (Atp1p in yeast). The suppressing activity of ATP1-111 requires intact beta (Atp2p) and gamma (Atp3p) subunits of mitochondrial ATP synthase, but not the stator stalk subunits b (Atp4p) and OSCP (Atp5p). ATP1-111 and other similarly suppressing mutations in ATP1 and ATP3 increase the growth rate of wild-type strains lacking mitochondrial DNA.

Yme2p is a mediator of nucleoid structure and number in mitochondria of the yeast Saccharomyces cerevisiae.

A large number of gene products have been identified that either directly or indirectly alter the inheritance of mitochondrial DNA. In yeast, we have used a unique genetic screen based on the transfer of DNA from mitochondria to nucleus to identify nuclear-encoded gene products that are targeted to mitochondria and impact the stable inheritance of mitochondrial DNA. A specific allele of one of these genes, yme2-4, prevents even the low wild-type rate of mitochondrial DNA transfer to the nucleus and imparts significant temperature-sensitive and respiratory-growth defects.

Formation of an energized inner membrane in mitochondria with a gamma-deficient F1-ATPase.

Eukaryotic cells require mitochondrial compartments for viability. However, the budding yeast Saccharomyces cerevisiae is able to survive when mitochondrial DNA suffers substantial deletions or is completely absent, so long as a sufficient mitochondrial inner membrane potential is generated. In the absence of functional mitochondrial DNA, and consequently a functional electron transport chain and F(1)F(o)-ATPase, the essential electrical potential is maintained by the electrogenic exchange of ATP(4-) for ADP(3-) through the adenine nucleotide translocator.

Mitochondrial DNA impacts the morphology of mitochondrial compartments.

Mitochondrial compartments of the yeast Saccharomyces cerevisiae experience continual morphological alterations. Mitochondrial compartments of wild-type yeast, when observed using fluorescent markers, are usually found to be a network of extended tubular structures. However, a quantitative analysis of mitochondrial structures in a genetically homogenous population of wild-type yeast revealed that although the majority of individual yeast cells contained the expected extended network of mitochondrial tubules, a significant number of cells were found to exclusively contain condensed globular mitochondrial compartments or a mixture of extended and globular mitochondrial compartments.

Genetic and biochemical basis for viability of yeast lacking mitochondrial genomes.

Yme1p, an ATP-dependent protease localized in the mitochondrial inner membrane, is required for the growth of yeast lacking an intact mitochondrial genome. Specific dominant mutations in the genes encoding the alpha- and gamma-subunits of the mitochondrial F(1)F(0)-ATPase suppress the slow-growth phenotype of yeast that simultaneously lack Yme1p and mitochondrial DNA. F(1)F(0)-ATPase activity is reduced in yeast lacking Yme1p and is restored in yme1 strains bearing suppressing mutations in F(1)-ATPase structural genes.

Maintenance of mitochondrial morphology is linked to maintenance of the mitochondrial genome in Saccharomyces cerevisiae.

In the yeast Saccharomyces cerevisiae, certain mutant alleles of YME4, YME6, and MDM10 cause an increased rate of mitochondrial DNA migration to the nucleus, carbon-source-dependent alterations in mitochondrial morphology, and increased rates of mitochondrial DNA loss. While single mutants grow on media requiring mitochondrial respiration, any pairwise combination of these mutations causes a respiratory-deficient phenotype. This double-mutant phenotype allowed cloning of YME6, which is identical to MMM1 and encodes an outer mitochondrial membrane protein essential for maintaining normal mitochondrial morphology.

Expression of the Saccharomyces cerevisiae gene YME1 in the petite-negative yeast Schizosaccharomyces pombe converts it to petite-positive.

Organisms that can grow without mitochondrial DNA are referred to as "petite-positive" and those that are inviable in the absence of mitochondrial DNA are termed "petite-negative." The petite-positive yeast Saccharomyces cerevisiae can be converted to a petite-negative yeast by inactivation of Yme1p, an ATP- and metal-dependent protease associated with the inner mitochondrial membrane. Suppression of this yme1 phenotype can occur by virtue of dominant mutations in the alpha- and gamma-subunits of mitochondrial ATP synthase.

Mechanisms of mitochondrial DNA escape to the nucleus in the yeast Saccharomyces cerevisiae.

The transfer of organelle nucleic acid to the nucleus has been observed in both plants and animals. Using a unique assay to monitor mitochondrial DNA escape to the nucleus in the yeast Saccharomyces cerevisiae, we previously showed that mutations in several nuclear genes, collectively called yme mutants, cause a high rate of mitochondrial DNA escape to the nucleus. Here we demonstrate that mtDNA escape occurs via an intracellular mechanism that is dependent on the composition of the growth medium and the genetic state of the mitochondrial genome, and is independent of an RNA intermediate.

YNT20, a bypass suppressor of yme1 yme2, encodes a putative 3'-5' exonuclease localized in mitochondria of Saccharomyces cerevisiae.

Mutation of YME genes in yeast results in a high rate of mitochondrial DNA escape to the nucleus. The synthetic respiratory growth defect of yme1 yme2 yeast strains is suppressed by recessive mutations in YNT20. Inactivation of YNT20 creates a cold-sensitive respiratory growth defect that is more pronounced in a yme1 background and which is suppressed by yme2.

Escape of mitochondrial DNA to the nucleus in yme1 yeast is mediated by vacuolar-dependent turnover of abnormal mitochondrial compartments.

Inactivation of Yme1p, a mitochondrially-localized ATP-dependent metallo-protease in the yeast Saccharomyces cerevisiae, causes a high rate of DNA escape from mitochondria to the nucleus as well as pleiotropic functional and morphological mitochondrial defects. The evidence presented here suggests that the abnormal mitochondria of a yme1 strain are degraded by the vacuole. First, electron microscopy of Yme1p-deficient strains revealed mitochondria physically associated with the vacuole via electron dense structures.

Inactivation of YME2/RNA12, which encodes an integral inner mitochondrial membrane protein, causes increased escape of DNA from mitochondria to the nucleus in Saccharomyces cerevisiae.

Inactivation of the yeast nuclear gene YMe2 causes an increased rate of DNA escape from mitochondria to the nucleus. Mutations in yme2 also show genetic interactions with yme1, a second gene that affects DNA escape from mitochondria to the nucleus. The yme1 cold-sensitive growth phenotype is suppressed by yme2 mutations.

Biochemical and functional analysis of the YME1 gene product, an ATP and zinc-dependent mitochondrial protease from S. cerevisiae.

Inactivation of YME1 in yeast causes several distinct phenotypes: an increased rate of DNA escape from mitochondria, temperature-sensitive growth on nonfermentable carbon sources, extremely slow growth when mitochondrial DNA is completely absent from the cell, and altered morphology of the mitochondrial compartment. The protein encoded by YME1, Yme1p, contains two highly conserved sequence elements, one implicated in the binding and hydrolysis of ATP, and the second characteristic of active site residues found in neutral, zinc-dependent proteases. Both the putative ATPase and zinc-dependent protease elements are necessary for the function of Yme1p as genes having mutations in critical residues of either of these motifs are unable to suppress any of the phenotypes exhibited by yme1 deletion strains.

Escape and migration of nucleic acids between chloroplasts, mitochondria, and the nucleus.

The escape and migration of genetic information between mitochondria, chloroplasts, and nuclei have been an integral part of evolution and has a continuing impact on the biology of cells. The evolutionary transfer of functional genes and fragments of genes from chloroplasts to mitochondria, from chloroplasts to nuclei, and from mitochondria to nuclei has been documented for numerous organisms. Most documented instances of genetic material transfer have involved the transfer of information from mitochondria and chloroplasts to the nucleus.

Mutations in the mitochondrial ATP synthase gamma subunit suppress a slow-growth phenotype of yme1 yeast lacking mitochondrial DNA.

In Saccharomyces cerevisiae, inactivation of the nuclear gene YME1 causes several phenotypes associated with impairment of mitochondrial function. In addition to deficiencies in mitochondrial compartment integrity and respiratory growth, yme1 mutants grow extremely slowly in the absence of mitochondrial DNA. We have identified two genetic loci that, when mutated, act as dominant suppressors of the slow-growth phenotype of yme1 strains lacking mitochondrial DNA.

Mitochondrial morphological and functional defects in yeast caused by yme1 are suppressed by mutation of a 26S protease subunit homologue.

The absence of functional Yme1p, a putative ATP and zinc-dependent protease localized to mitochondria of yeast, results in abnormal mitochondrial function and morphology. Yeast lacking Yme1p lose DNA from mitochondria at an accelerated rate, fail to grow on nonfermentable carbon sources at 37 degrees C, and have severely deficient growth if mitochondrial DNA suffers large deletions or is completely lost. In place of the normal reticulated mitochondrial network, strains lacking Yme1p have punctate mitochondria with some grossly swollen compartments.

AFG2, an essential gene in yeast, encodes a new member of the Sec18p, Pas1p, Cdc48p, TBP-1 family of putative ATPases.

A gene from Saccharomyces cerevisiae was sequenced that encodes a protein with homology to a family of putative ATPases. These homologous proteins include the yeast cell division cycle protein Cdc48p and its mammalian homologues VCP and p97; Sec18p and its mammalian homologue NSF, proteins necessary for fusion of transport vesicles to target membranes in the secretory pathway; Pas1p, a protein necessary for peroxisome biosynthesis in yeast; Yme1p, a yeast mitochondrial protein that influences the rate of DNA escape from mitochondria; and TBP-1, MSS1 and Sug1p, proteins that interact with transcription factors. This newly sequenced gene, named AFG2 for ATPase family gene, is located on chromosome XII 5' to the SLP1/VPS33 open reading frame and encodes an essential protein of 780 amino acids that is most homologous to Cdc48p.

Inactivation of YME1, a member of the ftsH-SEC18-PAS1-CDC48 family of putative ATPase-encoding genes, causes increased escape of DNA from mitochondria in Saccharomyces cerevisiae.

The yeast nuclear gene YME1 was one of six genes recently identified in a screen for mutations that elevate the rate at which DNA escapes from mitochondria and migrates to the nucleus. yme1 mutations, including a deletion, cause four known recessive phenotypes: an elevation in the rate at which copies of TRP1 and ARS1, integrated into the mitochondrial genome, escape to the nucleus; a heat-sensitive respiratory-growth defect; a cold-sensitive growth defect on rich glucose medium; and synthetic lethality in rho- (cytoplasmic petite) cells. The cloned YME1 gene complements all of these phenotypes.

Nuclear mutations in Saccharomyces cerevisiae that affect the escape of DNA from mitochondria to the nucleus.

We have inserted a yeast nuclear DNA fragment bearing the TRP1 gene and its associated origin of DNA replication, ARS1, into the functional mitochondrial chromosome of a strain carrying a chromosomal trp1 deletion. TRP1 was not phenotypically expressed within the organelle. However, this Trp- strain readily gave rise to respiratory competent Trp+ clones that contained the TRP1/ARS1 fragment, associated with portions of mitochondrial DNA (mtDNA), replicating in their nuclei.

Structural dynamics of the mitochondrial compartment.

The metabolic activities of mitochondria have been extensively characterized. However, there is much less known about the morphogenic changes of the mitochondrial compartment during growth, development and aging of the cell and the consequences of those structural changes on cellular metabolism. There is a growing body of evidence for interactions of mitochondria with cytoskeletal components and changes of mitochondrial structure during development and in response to changing environmental conditions.